Intrinsic factors and CD1d1 but not CD1d2 expression levels control invariant natural killer T cell subset differentiation

Invariant natural killer T (NKT) cell subsets are defined based on their cytokine-production profiles and transcription factors. Their distribution is different in C57BL/6 (B6) and BALB/c mice, with a bias for NKT1 and NKT2/NKT17 subsets, respectively. Here, we show that the non-classical class I-like major histocompatibility complex CD1 molecules CD1d2, expressed in BALB/c and not in B6 mice, could not account for this difference. We find however that NKT cell subset distribution is intrinsic to bone marrow derived NKT cells, regardless of syngeneic CD1d-ligand recognition, and that multiple intrinsic factors are likely involved. Finally, we find that CD1d expression levels in combination with T cell antigen receptor signal strength could also influence NKT cell distribution and function. Overall, this study indicates that CD1d-mediated TCR signals and other intrinsic signals integrate to influence strain-specific NKT cell differentiation programs and subset distributions.

have more NKT2/17 despite lower CD1d expression?However, the authors have gone further than the previous experiments to prove their points, and they have created the appropriate genetic models for testing CD1d1 and CD1d2 function.Furthermore, the experiment in Fig. 4 indicates that not only is the level of CD1d expression not governing for determining the NKT cell subset frequency but also that B6 and BALB positively selecting double positive thymocytes are not presenting different ligands that induce differential subset representations.Why do the authors refer to CD1d2 in B6 as a pseudogene?Their data and previous research (ref 29) show that B6 CD1d2 can select NKT cells.The data showing increased Vb7 in CD1d+/-mice was shown originally by MacDonald (PMID 16455960).staining for some BALB/c and B6 subsets.Do the authors have an explanation or speculation?Line 439: "In fact, the subsets skewed toward Vb7 expression have a developmental advantage (NKT2 or NKT1), whereas the subset with the lowest Vb7 expression (NKT17) has a reduced developmental potential."How was developmental potential measured?

Reviewer #2 (Remarks to the Author):
In this ms, Amable and colleagues investigated the cues that drive NKT differentiation into NKT1, NKT2, and NKT17 subsets after agonist selection in the thymus.This is an exciting and complete piece of work and should be suitable for the readership of natcomms.The authors thoroughly investigated the role of CD1d1 and CD1d2 isogenes for thymic NKT differentiation and concluded that CD1d2 expression in Balb/c but not in B6 is not the reason for an NKT2 bias in Balb/c.To this end, they generated invividual KO mice for the two CD1d isoforms.Strong evidence for rather CD1d-unrelated intrinsic factors driving the distinct differentiation of B6 vs. Balb/c NKT cells is presented in mixed BM Xmera in Fig. 4. Next, they use elegant haploinsufficient mice to show that CD1d1 expression levels still influence the differentiation of NKT0 into NKT17 cells I would suggest that the title ("Intrinsic factors control …") is maybe a bit misleading and could be adapted, as the data shown indicate that rather "Intrinsic factors and CD1d1 but not CD1d2 expression levels control NKT differentiation into NKT1, NKT2, and NKT17 subsets".Also, it would be nice to discuss candidates for such intrinsic factors that favor NKT2 in Balb/c.Do these factors also promote type-2 innate MAIT and gd T cell development in Balb/c or is this specific for NKT differentiation?Very minor: The call-out for We thank both reviewers for their constructive comments.
Reviewer #1 (Remarks to the Author): Why do the authors refer to CD1d2 in B6 as a pseudogene?Their data and previous research (ref 29) show that B6 CD1d2 can select NKT cells.
In WT B6 mouse strain, we refer to CD1d2 as a pseudogene because in this strain the gene harbors a frameshift mutation at the beginning of the fourth exon encoding the a3 domain of CD1D2.This mutation is predicted to abolish its surface expression (The Journal of Immunology, 1998, 160: 3128-3134).Cd1d2 is therefore considered a pseudogene in B6 WT mice.This frame shift mutation does not exist in 129/sv and BALB/C strains, where Cd1d2 is not a pseudogene.We agree with the referee that in our study and in the study by Gapin's group (ref 29), the results indicate that B6 CD1d2 can nevertheless select NKT cells.However, it should be kept in mind that these mice are CD1d1deficient mice expressing CD1d2 as they were generated with ES cells from the 129/SV mouse strain expressing CD1d2.

The data showing increased Vb7 in CD1d+/-mice was shown originally by MacDonald (PMID 16455960).
The referee is correct, and we have now mentioned in the discussion that our results are in agreement with those of MacDonald's study.In addition, our results show that the increase in Vb7 levels differs between NKT cell subsets (Fig. 5E).

Fig. 1B and elsewhere. Are the NKT cells in BALB/c mice larger in size? Should the MFI be corrected for a cell size difference?
NKT cells in BALB/c mice are not larger in size and thus there is no need to correct MFI for cell size.We have added this information to supplemental figure S1 (S1C)., b, and c) and NKT2 (NKT2a, and b).It also showed that NKT2 cells comprise precursors to NKT1 and NKT17 cells.We thus speculate that the bi-modal staining pattern observed with our cells could be due to the heterogeneity of NKT cell subsets.

Some of the flow cytometry measurements indicate populations have a bi-modal staining pattern. Some examples include
Line 439: "In fact, the subsets skewed toward Vb7 expression have a developmental advantage (NKT2 or NKT1), whereas the subset with the lowest Vb7 expression (NKT17) has a reduced developmental potential."How was developmental potential measured?
We are sorry about the confusion induced by this passage, as we did not measure any developmental potential.The sentence has now been replaced by the following one in line 339: "Our results show that V7 use correlates with changes in NKT2 and NKT17 but not NKT1 cell frequencies in CD1d+/mice.In fact, the subsets skewed toward V7 expression have an unaltered (NKT1) or increased (NKT2) frequency, whereas the subset with the lowest V7 expression (NKT17) has a reduced cell frequency".
Reviewer #2 (Remarks to the Author): We took this advice into consideration and changed the title as suggested.
Also, it would be nice to discuss candidates for such intrinsic factors that favor NKT2 in Balb/c We have answered this question based on newly obtained results that we correlated with data from the literature.Below is a short version of the detailed discussion from lines 434 to 503, and in supplemental figure S5.
To address this question, we analyzed the expression of intrinsic factors related to the development and/or function of NKT1 (T-bet, IL-15R), NKT2 (IL-25R), and NKT17 (RORt, IL-7R, TGFbRII, phospho-SMAD2/3).We found no differences in their expression levels that could explain differential distribution of NKT cells in B6 vs BALB/c mice (data provided below in Figure 1 to the reviewers).However, we found that GATA3 was expressed at two-fold higher levels in NKT2 cells from BALB/c compared to cells from B6 mice (Supp Fig. S5A).Similarly, our results in figure Fig. 1C  We have corrected the order of appearance of the figures in the text

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): This revised manuscript described experiments designed to address why the functional subsets of thymic natural killer T (NKT) cells differ greatly between B6 mice, which have mostly Th1-like or NKT1 cells, compared to BALB/c, with more NKT2 cells.The manuscript is improved in some respects by the author's response, and it establishes that NKT cell precursor intrinsic factors are responsible.They have added new data showing increased expression of GATA-3 in BALB/c, and decreased Ezh2 in BALB/c NKT thymus subsets.
Based on B6 strain genetic alterations, these were predicted to increase NKT2 cells.They also found increased Nrp2 expression in BALB/c, based on data mining, but did not do experiments to validate its importance.This section belongs in the results, and it is poorly written, with several references to "data not shown," which is no longer acceptable for most journals, and discussion of experiments they might do in the future.The antibody staining for Nrp2 is not properly described in the text, and the increase in H3K27me3 in BALB/c NKT cells does not agree with what is written.
Some data from other labs and data presented here suggest that decreased TCR affinity during selection will promote NKT1 (or conversely, that NKT2 cells have obtained an increased TCR signal).If this is the case, then why don't CD1d+/-mice, which presumably have a decreased signal, also have fewer NKT2 cells?Sorry that this point was not raised earlier by this reviewer.
Minor issues Use of scientific notation for the numbers in Fig 1A Line 139.Is the CD1d2 TG mice on the B6 or CD1d1-/-background?
Line 422 needs a slight clarification to note that while NKT1 cells are increased in the BALB/c chimeras compared to BALB/c mice, there is still a higher frequency of NKT2 in the chimeras than in B6 mice.

Reviewer #2 (Remarks to the Author):
The authors did a great job in thoroughly replying to all the two reviewers' concerns.The ms should now be very suitable for publication in NCOMMS.However, I am not sure if "data not shown" on page 21 is supported by the journal's policy.
Fig. 1B and elsewhere.Are the NKT cells in BALB/c mice larger in size?Should the MFI be corrected for a cell size difference?Some of the flow cytometry measurements indicate populations have a bi-modal staining pattern.Some examples include Fig S1A PLZF staining for B6 NKT2 and Fig S1B SLAMF6 Fig 5f is missing and Fig 5g is mentioned after Fig 6.
Fig S1A PLZF staining for B6 NKT2 and Fig S1B SLAMF6 staining for some BALB/c and B6 subsets.Do the authors have an explanation or speculation?Baranek et al. (Cell Reports 32, 108116, September 8, 2020) performed an unbiased single-cell transcriptomic analysis (scRNA-seq) on thymic NKT cells.This study revealed discrete subsets among NKT1 (NKT1a and Supp Fig.S1Dreveal higher expression of SLAM proteins at the surface of NKT2 cells from BALB/c compared to B6 mice.Both GATA3 and SLAM-associated proteins (SAP) have been described to favor NKT2 cell development(J Immunol, 2006, 177 : 6650-6659 ; Eur J Immunol, 2016, 46: 2162-2174), their higher expression in BALB/c NKT2 cells could contribute in part to the predominance of this subset in these mice.In addition, we exploited a dataset made available by Georgiev et al.(Nat Commun, 2016, 7 : 13116)   following their analysis of transcriptional expression of NKT cell subsets in B6 vs BALB/c mice.We found EZH2 transcript expression to be higher in B6 NKT2 cells compared to BALB/c NKT cells (Supp Fig.S5B).Our intracellular staining analysis showed a higher H3K27me3 level in B6 NKT2 cells compared to BALB/c NKT cells (Supp Fig.S5C).Based on a study showing that loss of Ezh2 and H3K27me3 in NKT cells favored NKT2 cell development (J Exp Med,2015, 212 : 297-306), its reduced level in BALB/c NKT cells could contribute in part to the higher NKT2 cell frequency in BALB/c mice.By further exploiting the Georgiev et al. dataset (NatCommun, 2016, 7 : 13116), we found NRP2 transcripts (encoding for neuropilin-2) to be highly expressed in BALB/c NKT2 cells compared to B6 mice (Supp Fig.S5D).We confirmed this increased expression by immunohistochemistry using an IL-4/GFP-enhanced transcript (4Get) strain, which expresses a fluorescent reporter in IL-4-producing cells, to visualize NKT2 cells (Supp Fig.5E).NRP2 mainly binds to semaphorin 3F and 3C (Sema3F, 3C) (Front Immunol, 2017, 8 :1228).In humans, the NRP2/Sema3F axis is reported to inhibit thymocyte migration in response to S1P1, a chemokine with a well-documented role in thymocyte egress from the thymus (PLoS One, 2014, e103405).Because BALB/c NKT2 cells express NRP2, this may render emigration of NKT2 inefficient in this strain, providing a potential explanation as to why NKT2 cells are more frequent in BALB/c mice.Future studies using NRP2-deficient mice will allow the functional consequences of NRP2 expression in BALB/c NKT2 cells to be verified.Based on these considerations, our results thus indicate that the intrinsic factors involved in the differential strain subset distribution are likely be multiple, including transcription factors, downstream signaling cascades, epigenetic factors, and migratory factors, among others.The fine regulation of these intrinsic factors can be linked to gene polymorphisms and/or regulatory enhancer/silencer regions.To uncover new genes controlling NKT cell differentiation and strain distribution, we are planning to deploy an unbiased genetic approach, using a collection of mice (CC collection)(Nat Genet,2004, 36 :1133-1137).This collection will allow us to correlate NKT cell subset distribution with genetic controls.Preliminary results show a variability in the distribution of NKT2 cells in the 8 founders and the 35 strains of the CC collection analyzed so far, supporting the use of this approach to search for genetic factors controlling NKT cell subset differentiation and distribution (data not shown).Do these factors also promote type-2 innate MAIT and gd T cell development inBalb/c or is this specific for NKT differentiation?These factors could potentially also promote type-2 MAIT and  T cell development in BALB/c mice.In fact, in addition to being important for NKT2 cells development (J Immunol, 2006, 177 : 6650-6659 ), GATA3 is also important for Tγδ2 development; these cells were efficiently depleted in Gata3 cKO mice (Nature communications, 2020, 11 : 18155-8).Within the  T cell compartment, it was also previously demonstrated that development of the T2 V1V6.3 (NKT) subset requires SAP (Journal of Immunology, 2010, 84: 6746-6755), indicating that SLAM/SAP signaling during development contributes to the emergence of NKT cells, MAIT cells, and at least the T2 T cell subset.Very minor: The call-out for Fig 5f is missing and Fig 5g is mentioned after Fig 6.
would suggest that the title ("Intrinsic factors control …") is maybe a bit misleading and could be adapted, as the data shown indicate that rather "Intrinsic factors and CD1d1 but not CD1d2 expression